This invention relates to synthetic cationic lipid compounds, liposome formulations and the use of such compounds via lipofection to introduce functional bioactive agents/biologically active substances to cultured cells.
Lipofection (or transfection), a process of introducing functional nucleic acids into cultured cells by using positively charged liposomes, was first described by Philip Felgner et al. a decade ago, and later shown, by K. L. Brigham, as applicable in vivo to experimental animals. See, Felgner et al., Proc. Natl. Acad. Sci. USA, 84, 74113-7417 (1987) and K. L. Brigham et al., Am. J. Med. Sci., 298, 278-281 (1989). Cationic lipids have become an increasingly important tool for many in vitro gene transfer applications, including several recent human gene therapy trials. As non-viral, synthetic DNA carriers, cationic lipids are particularly attractive because they are non-immunogenic, simple to use, can deliver DNA of a broad range size, and can be manufactured in large quantity. Although lipofection is quite efficient in vitro under serum-free conditions, its use in vivo when delivered as DNA/lipid complex, lipoples, via intravenous or airway routes, was limited to the presence of proteins and polysaccharides in the body fluids and mucus that strongly inhibit the transfection efficiency. Recent efforts to search for more efficient lipids and/or improved DNA/liposome formulations have resulted in dramatic increases of in vivo transfection efficiency. See, Solodin, I. et al., Biochemistry, 34, 13537-13544 (1995); Templeton, N. S., et al., Nat. Biotech., 15(7), 647-652 (1997); Thierry, A. R. et al., Proc. Natl. Acad. Sci. USA, 92, 9742-9746 (1995); Li, S. et al., Gene Ther., 4, 891-900 (1997); Liu, Y., et al., Nat. Biotechnol., 15(2), 167-173 (1997); Liu, F. et al., Gene Ther., 4(6), 517-523 (1997); and Song Y. K., et al., Hum. Gene Ther., 8, 1585-1594 (1997).
Using a few well known cationic lipids, researchers have carefully studied key parameters that affect transfection efficiency of intravenously administered lipoplex in mouse. See, also Hong K., et al., FEBS Letters, 400, 233-237 (1997). These studies have shown that a 2-16 fold excess of cationic liposome over DNA were necessary for high level gene expression in lungs and other organs, liposomes composed of 1,2, -dioleyl-3-N,N,N-trimethyl amino propane chloride (DOTMA) were significantly more active than that composed other cationic lipids. See, Song et al. Liposomes prepared from pure cationic lipid, such as DOTMA or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or mixtures of a cationic lipid and cholesterol or Tween 80 could mediate more efficient gene transfer than those formulations composed of a cationic lipid and a neutral phospholipid 1,2-dioleoyl-sn-glycero-3-phosphotidylethanolamine (DOPE) and the lipoplex prepared from extruded multilamellar liposomes were more active than those prepared from sonicated small unilamellar liposomes. See, Hong et al., Liu, Y et al., Liu, F. et al, Song et al., and Li, S. et al. With these improvements, the level of reporter gene expression in lungs after intravenous administration of optimized lipoplex were estimated 100 to 1000 fold more efficient than those prepared according to previously reported studies using DOTMA/DOPE liposomes and less lipid to DNA ratios. However, the improvements were also associated with noticeable toxicity of varying degrees of lipid/DNA treated animals due to toxic effect of large excess of cationic lipids or lipoplexes themselves. Therefore, searching for cationic lipids with less toxicity and formulations that function at reduced lipid to DNA ratios seem to be reasonable approaches to the problem of lipofection-related toxicity. We report the synthesis of a novel series of cationic lipids and test of their in vivo transfection activity in mice.
High level transfection in vivo in lungs and several other organs using lipoplex have been reported by a number of groups, as cited in the text above. One of the conclusions drawn from these studies is that relatively high lipid to DNA ratios is required to achieve high levels of transfection in vivo (Hong, K., et al., Liu, Y., et al., Liu, F., et al., and Li, S., et al.) Besides the high charge ratios, other factors such as the use of cholesterol, instead of DOPE as helper lipid (Hong, K., et al., Templeton, N. S., et al., Liu, Y., et al, Song, Y. K., et al. and Li, S., et al); multilamellar liposomes of about 200 nm in size rather than small unilamellar liposomes (Liu, Y., et al.) and the use of polycations and polymers in the cationic lipid-DNA complexes also contribute to the high level transfection (Hong, K., et al. and Li, S., et al.). The least mentioned factor in these studies was the side effects that are associated with these high level transfections in vivo.
It is therefore an object of the present invention to provide cationic lipids which are less toxic in pharmaceutical formulations and function at reduced lipid to DNA ratios than existing cationic lipids.
It is also an object of the present invention to provide a liposome, with or without a helper lipid, which is less toxic than prior art liposomes.
It is also an object of the present invention to provide cationic liposome pharmaceutical formulations which enhance intracellular delivery of DNA to a less toxic extent than previously described lipophilic compounds.
It is also an object of this invention to provide a lipoplex that has a transfection activity which is higher than transfection activity of the prior art liposome.
It is also and object of this invention to provide improved lipid and liposome formulation for treating a disease in a mammal via transfection.
It is a further object of the present invention to provide cationic liposome formulations which demonstrate superior efficacy.
In accordance with the purpose(s) of this invention, as embodied and described herein, this invention, in one aspect, relates to novel cationic lipids, and their use in pharmaceutical formulations for the intra cellularly delivery of bioactive agent.
One aspect of the invention relates to a compound of the general formula I: 
or a pharmaceutically acceptable salt or ester thereof; wherein:
R1 and R2 are the same or different and are from C6 to C24 alkyl or aryl;
Y and Z are both xe2x80x94Oxe2x80x94C(O)xe2x80x94 or xe2x80x94Oxe2x80x94; 
or xe2x80x94C(O)xe2x80x94Oxe2x80x94; and
n=1-6.
Another aspect of the invention relates to a compound of formula I(a): 
or a pharmaceutically acceptable salt or ester thereof, wherein:
R1 and R2 are independently from C6 to C24 alkyl or alkenyl or one of R1 or R2 is C6 to C24 alkyl or alkenyl and the other is absent, or R1 and R2 are independently aryl;
Y and Z are the same and are xe2x80x94Oxe2x80x94C(O)xe2x80x94, or one of Y and Z is xe2x80x94Oxe2x80x94C(O)xe2x80x94 and the other is xe2x80x94OH; 
R3 is C1-6 alkyl, aryl, aryloxy, alkene, or a protecting group; and
n=1-6.
Another aspect of the invention relates to a compound of formula I(b): 
or a pharmaceutically aceptable salt or ester thereof, wherein:
R1 and R2 are the same or different and are from C6 to C24 alkyl or alkenyl or aryl;
Y and Z are both xe2x80x94Oxe2x80x94C(O)xe2x80x94;
A is xe2x80x94C(O)xe2x80x94Oxe2x80x94;
R4 is C1-6 alkyl;
a=0 or 1;
n=1-3;
q=0-3;
Xxe2x88x92 is a halogen anion or is absent; and
the S and N atoms each independently have a positive charge or the positive charge is absent.
The novel N-[(2,3-dioleoyloxy)propyl]-L-lysinamide (LADOP) cationic lipid has lysine as a head group and two long fatty chains as lipid anchors linked through biodegradable diester and amide bonds. Liposome/DNA complexes prepared with a plasmid containing a luciferase reporter gene delivered either intravenously or intratracheally to mice demonstrated high level expression of the transgene. The magnitude of transgene expression was related to the ratio of lipid to DNA; high level of transgene expression occurred when lipid/DNA charge ratios reached 1:8 to 1:24. The duration of the transgene expression in lungs of mice treated with liposome/DNA complex delivered intravenously was transient, disappearing by 48 hours after transfection. However, a second intravenous transfection at 48 hours after the first injection resulted in almost identical gene expression and duration.
The LADOP cationic lipid is a very efficient molecule as a result of careful design. The overall structure offers a bilayer-forming lipid with good membrane fluidity. Therefore, the novel lipid can actively transfer genes without a commonly required helper lipid, DOPE. LADOP has a stable amide bond linkage between the head group and the lipid anchor to ensure stability in solution. It has two cationic charges, while lysl-PE has only one net charge per molecule. The diester bonds in LADOP render the lipid biodegradable. This feature has been demonstrated in our in vivo studies in which the animals tolerated repeated intravenous doses of DNA/LADOP complex well and transgene expression patterns of the first injection and second injection shortly after were identical both in magnitude and duration. In two recent reports, a twenty day interval between treatments was needed for effective repeat administration, using DOTMA and DOTAP, two formulations that have been widely used. The fact that the novel LADOP cationic compound mediated efficient transfection both intravenously and intratracheally, together with features such as simplicity of synthesis and formulation and minimal toxicity, make this a very attractive and useful reagent for in vivo gene delivery, and ultimately, gene therapy.
Another aspect of the invention relates to lysine amide cationic compounds of the general formula III: 
or a pharmaceutically acceptable salt or ester thereof,
wherein:
R1 and R2 are the same and are from C6-24 alkyl or alkenyl; and
Q is a cationic charged head group.
The novel cationic lipid is a lysine amide cationic lipid. This novel cationic lipid has long fatty chains and a cationic charged head group which can be categorized into three groups based on the chemical structures of the head group. The three different head groups represented by formula II are 1) bis-imidoamide head groups; 2) head groups having two to 8 amino groups generated from propylamino repeat units; and 3) head groups having varied numbers of lysl residues linked by amide bonds through alpha or epsilon amino groups in asymmetric and symmetric configurations.
These new cationic lipids are micelle-forming lipids in their salt form and have excellent DNA condensation abilities and capability of forming small complexes with DNA. In vitro test revealed that most of these lipids have high transfection activity on cells in general. However, the two lipids belonging to group three, with three to five lysine groups symmetrically and spaciously distributed over the molecules are 2 to 5 times more potent than those lipids with similar numbers of lysine groups distributed in dendritic (tree) shape or linear pattern. These novel compounds clearly demonstrate the important relationship of structure and transfection activity of a cationic lipid.
Another aspect of the invention relates to cationic lipid compounds of formula II: 
or a pharmaceutically acceptable salt or ester thereof,
wherein:
R1 and R2 are the same or different and are from C6 to C24 alkyl or alkenyl;
Y and Z are both xe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94C(O)xe2x80x94; or when one of Y or Z is xe2x80x94Oxe2x80x94C(O)xe2x80x94, the other is xe2x80x94Oxe2x80x94;
R3 is C1-6 alkyl or alkene, aryloxy, aryl, a protecting group, or is absent;
R6 and R7 are taken together with the N atom to form a 5 to 8-membered heterocyclic ring;
Xxe2x88x92 is a halogen anion or is absent; and
the N atom has a positive charge or is neutral.
Another aspect of the invention relates to a compound of formula IIa: 
or a pharmaceutically acceptable salt or ester thereof,
wherein:
R1 and R2 are the same or different and are from C1 to C23 alkyl or alkenyl, aryl or heterocyclic;
R6 and R7 are taken together with the N atom to form a 5 to 8-membered heterocyclic ring; and
Xxe2x88x92 is a halogen anion.
Another aspect of the invention relates to a compound of formula (IIb): 
R1 and R2 are the same or different and are from C6 to C24 alkyl or alkenyl or aryl;
Y and Z are both xe2x80x94Oxe2x80x94 or xe2x80x94Oxe2x80x94C(O)xe2x80x94;
R6and R7 are taken together withthe N atom to form a 5 to 8-membered heterocyclic ring in which the heterocyclic N is unsubstituted or substituted with one C1-3 alkyl groups;
A is xe2x80x94Cxe2x80x94(O)xe2x80x94Oxe2x80x94;
n=1-6; and
Xxe2x88x92 is a halogen anion or is absent.
Another aspect of the invention relates to the following compounds represented by: 
Another aspect of the invention relates to the following compounds represented by: 
The novel cationic compound is a heterocyclic cationic lipid. Included within this aspect of the invention are several monovalent double chain cationic lipids. Namely, 3-N-methyl-1,2-dioleylpropandiol morpholino chloride (MMET) and 3-N-methyl-1,2-dioleoylpropandiol morpholino chloride (MMES). The novel cationic lipids represented by formula III has a simple ammonium or an ammonium located at the base of a ring structure as a head group which is linked to two long, mono-unsaturated fatty chains as lipid anchors. These lipids contain several types of bonds which link the head groups and lipid anchors, including biodegradable diester bonds, stable diether bonds, alkyl bonds or mixed bonds.
The liposomes composed of a single cationic lipid, or a mixture of cationic lipid and non-charged helper lipids, such as cholesterol at 1:1 ratio, were prepared and used to complex DNA at charge ratios of from 0.75-16xc2x1. Liposome/DNA complexes prepared with a plasmid containing a luciferase reporter gene were administered intravenously to mice. After 12 hours, luciferase activity was demonstrated at high levels in lung, liver, spleen and other organs. The magnitude of transgene expression was related the ratio of lipid to DNA and was determined by the lipid composition and the type of helper lipids. For liposomes prepared from single cationic lipids, transgene expression occurred when lipid/DNA ratios reached 1:4 and peaked at ratio of 1:16. Among these lipids, the transfection activity of the compound MMET was much higher than the prior art liposomes composed of DOTMA and/or DOTAP or DOTMA-cholesterol. These prior art complexes caused toxicity to the mice, especially at higher doses.
Another aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
The novel cationic compound is a benzyl compound. The novel cationic lipid represented has a benzyl group attached to a nitrogen atom, which is linked to two long mono-unsaturated fatty chains as lipid anchors. These lipids contain several types of bonds which link the head groups and lipid anchors, including biodegradable diester bonds, diether bonds, alkyl bonds or mixed bonds.
Another aspect of the relates to cationic compounds having the formulas:
chol=cholesteryl group
xe2x80x83Polyethyleimine-cholxe2x80x83xe2x80x83C-7
Dendrimer-0-cholxe2x80x83xe2x80x83C-8
xe2x80x83Dendrimer-1-cholxe2x80x83xe2x80x83C-9

or a pharmaceutically acceptable salt or ester thereof.
The novel cationic compound is a cholesterol (or chlolesteryl) containing cationic lipid.
Another aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutical acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to a compound of formula IV: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to a compound of formula (V): 
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
Another aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
One aspect of the invention relates to compounds having the formulas: 
or a pharmaceutically acceptable salt or ester thereof.
Each of these cationic lipids may be used in pharmaceutical formulations in the form of liposomes with a bioactive agent/biologically active agent either alone or in combination with other lipid substituents.
In another aspect, the invention relates to pharmaceutical compositions comprising the aforementioned inventive compounds in combination with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include aqueous solutions and complex delivery systems as further described herein. Preferably, the pharmacologically acceptable carrier is a liposome.
The invention also encompasses pharmaceutically acceptable esters, and salts of such compounds, as will be explained in detail, infra.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.